Project Summary Abstract Motor skill learning, like learning how to speak, walk, or play a musical instrument, is an essential part of behavioral development. Extensive past work has characterized motor learning at psychological, neurophysiological, and cellular levels. However, there is relatively poorer understanding of how the neural systems that underlie this form of learning change at the molecular level during behavioral plasticity. Moreover, there is little definition of how plasticity within a multi-locus neural system is reflected in coordinated alterations to gene expression. Such a systems-level understanding of the interconnections between behavioral and molecular plasticity is essential to broader understanding of the pathological alterations in neurological disease and the molecular conditions that promote or constrain plasticity. In particular, song learning is highly analogous to speech learning in humans and to general motor skill acquisition, exhibiting similar behavioral trajectories and employing homologous neural systems. Disruptions to these neural systems in humans are strongly associated with motor-associated neurodegenerative disease such as Parkinson's and Huntington's diseases. This proposal will use song learning in songbirds, a tractable and ethologically relevant model of motor skill learning with highly controllable inputs, precisely measurable outputs, and defined neural substrates, to characterize the transcriptional states that underlie motor skill acquisition and destabilization. The broad, long-term objective of this work is to understand the molecular mechanisms that contribute to plasticity in a complex learned behavior, both during normal development and during pathological disruption. This proposal will focus on two specific hypotheses. The first aim will test the hypothesis that song acquisition in juveniles induces specific transcriptional responses in the song system. Moreover, this aim will test the hypothesis that initial tutor song exposure and subsequent song learning induce distinct transcriptional states. The second aim will test the hypothesis that deafening in adult birds, a manipulation that drives song destabilization, drives transcriptional responses in the song system. Past work has demonstrated that deafening-induced song destabilization requires intact signaling from the cortical-basal ganglia system that underlies song learning. Following from this result, this aim will also test the hypothesis that this dependency extends to the level of gene expression by examining transcriptional responses in the song system in deafened birds that lack intact cortical-basal ganglia output to the song motor pathway. To enable this work, we have developed a low-cost and high-throughput RNA sequencing method that permits the analysis of gene expression in hundreds of individual laser microdissected samples from single animals. Together, these experiments will establish a framework for understanding the molecular basis of alterations in complex behaviors.